Deciphering the role of Epstein-Barr virus in the pathogenesis of T and NK cell lymphoproliferations

Epstein-Barr virus (EBV) is a highly successful herpesvirus, colonizing more than 90% of the adult human population worldwide, although it is also associated with various malignant diseases. Primary infection is usually clinically silent, and subsequent establishment of latency in the memory B lymphocyte compartment allows persistence of the virus in the infected host for life. EBV is so markedly B-lymphotropic when exposed to human lymphocytes in vitro that the association of EBV with rare but distinct types of T and NK cell lymphoproliferations was quite unexpected. Whilst relatively rare, these EBV-associated T and NK lymphoproliferations can be therapeutically challenging and prognosis for the majority of patients is dismal. In this review, we summarize the current knowledge on the role of EBV in the pathogenesis of these tumours, and the implications for treatment. \ud \u

Biochemical and virologic analysis of the human cytomegalovirus encoded UL97

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in immunocompromised patients. It is one of the most important opportunistic agents in the pathology of HFV, where it frequently causes sight-threatening retinitis. Currently approved antiviral drugs include ganciclovir, cidofovir and foscarnet. The HCMV UL97 gene encodes a phosphotransferase that has been shown to catalyse the initial phosphorylation of ganciclovir (GCV) to its monophosphate form, whereafter it is converted to the active triphosphate by cellular kinases. HCMV disease often necessitated long-term antiviral therapy, which in turn led to the emergence of ganciclovir resistant strains of HCMV. Mutations in the UL97 gene have been shown to impair GCV phosphorylation, thus conferring GCV resistance to HCMV and have been associated with disease progression. HCMV has been observed in almost every organ system in the body, but little information is available on the extent of UL97 mutations in the organs of chronically treated AIDS patients. A point mutation assay was employed to determine the prevalence of UL97 mutations in post mortem organs of patients who died with AIDS. The results showed that quantitative differences in resistant genotypes between organs of the same individual and between individuals occurs which has relevance to the management of patients on long term therapy for HCMV. In order to investigate functional effects of UL97 drug resistance mutations, wild type and mutant UL97 proteins were produced using a recombinant baculovirus expression system. Autophosphorylation properties of wild type and mutant UL97 were similar, while the mutations reduced ganciclovir phosphorylation to below 20% of the wild type UL97. The novel antiviral benzimidavir, inhibited autophosphorylation of wild type and mutant UL97 proteins. Enzyme kinetic analysis demonstrated benzimidavir acts by competitive inhibition of ATP binding to UL97. The mutation at codon 397, which confers resistance to benzimidavir, reduced autophosphorylation by 90%. These observations have implications for combination therapy with benzimidavir and ganciclovir

From pathobiology to targeted treatment in Epstein Barr virus related T cell and Natural Killer cell lymphoproliferative diseases

Epstein Barr virus (EBV), a gamma-1 herpesvirus, establishes a life-long latent infection in the majority of individuals worldwide. Whilst this seemingly innocuous virus is carried as an asymptomatic infection of memory B cells, EBV is paradoxically linked to two pre-malignant lymphoproliferative diseases (LPDs) and up to nine aetiologically distinct human tumours, accounting for up to 200,000 cancers per year. Although these malignancies primarily occur in B cells and epithelial cells, correlating with the natural tropism of the virus, on rare occasions EBV also infects T and NK cells leading to various pre-malignant LPDs, lymphomas and leukaemia. These conditions are often highly aggressive and are associated with hyperinflammation and organ dysfunction leading to substantial mortality. While, in recent years improvements in chemotherapy, especially asparaginase based regimens, have led to improved outcomes for some, these conditions are often intrinsically chemoresistant with allograft often providing the only curative option. In this review, we address the impact of genetic and epigenetic changes on the pathogenesis of the T/NK cell LPDs and bring together an analysis of recent clinical trials attempting to target these processes. We describe the main molecular characteristics across the range of EBV-associated T/NK cell diseases, from somatic mutations effecting epigenetic regulators seen across EBV related T/NK LPD subtypes to the marked dysregulation of the JAK-STAT-MYC axis observed in higher grade disease such as extranodal NK/T lymphoma (ENKTL). We then go on to analyse the novel therapeutic options available including immunomodulation and small molecule inhibitors. While these approaches show promise in early phase trials there is still much scope for improvement and a need to better understand the pathophysiology to design rational combination treatments

Deciphering the roles of myeloid derived suppressor cells in viral oncogenesis

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of myeloid cells derived from monocyte and granulocyte precursors. They are pathologically expanded in conditions of ongoing inflammation where they function to suppress both innate and adaptive immunity. They are subdivided into three distinct subsets: monocytic (M-) MDSC, polymorphonuclear (or neutrophilic) (PMN-) MDSC and early-stage (e-) MDSC that may exhibit differential function in different pathological scenarios. However, in cancer they are associated with inhibition of the anti-tumour immune response and are universally associated with a poor prognosis. Seven human viruses classified as Group I carcinogenic agents are jointly responsible for nearly one fifth of all human cancers. These viruses represent a large diversity of species, including DNA, RNA and retroviridae. They include the human gammaherpesviruses (Epstein Barr virus (EBV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV), members of the high-risk human papillomaviruses (HPVs), hepatitis B and C (HBV, HCV), Human T cell leukaemia virus (HTLV-1) and Merkel cell polyomavirus (MCPyV). Each of these viruses encode an array of different oncogenes that perturb numerous cellular pathways that ultimately, over time, lead to cancer. A prerequisite for oncogenesis is therefore establishment of chronic infection whereby the virus persists in the host cells without being eradicated by the antiviral immune response. Although some of the viruses can directly modulate the immune response to enable persistence, a growing body of evidence suggests the immune microenvironment is modulated by expansions of MDSCs, driven by viral persistence and oncogenesis. It is likely these MDSCs play a role in loss of immune recognition and function and it is therefore essential to understand their phenotype and function, particularly given the increasing importance of immunotherapy in the modern arsenal of anti-cancer therapies. This review will discuss the role of MDSCs in viral oncogenesis. In particular we will focus upon the mechanisms thought to drive the MDSC expansions, the subsets expanded and their impact upon the immune microenvironment. Importantly we will explore how MDSCs may modulate current immunotherapies and their impact upon the success of future immune-based therapies.</p

The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein

BACKGROUND: The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required. METHODS: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities. RESULTS: Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM. DISCUSSION: Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients

Ocrelizumab B cell depletion has no effect on HERV RNA expression in PBMC in MS patients

Background: Epstein barr virus (EBV) infection of B cells is now understood to be one of the triggering events for the development of Multiple Sclerosis (MS), a progressive immune-mediated disease of the central nervous system. EBV infection is also linked to expression of human endogenous retroviruses (HERVs) of the HERV-W group, a further risk factor for the development of MS. Ocrelizumab is a high-potency disease-modifying treatment (DMT) for MS, which depletes B cells by targeting CD20. Objectives: We studied the effects of ocrelizumab on gene expression in peripheral blood mononuclear cells (PBMC) from paired samples from 20 patients taken prior to and 6 months after beginning ocrelizumab therapy. We hypothesised that EBV and HERV-W loads would be lower in post-treatment samples.Methods: Samples were collected in Paxgene tubes, subject to RNA extraction and Illumina paired end short read mRNA sequencing with mapping of sequence reads to the human genome using Salmon and differential gene expression compared with DeSeq2. Mapping was also performed separately to the HERV-D database of HERV sequences and the EBV reference sequence.Results: Patient samples were more strongly clustered by individual rather than disease type (relapsing/remitting or primary progressive), treatment (pre and post), age, or sex. Fourteen genes, all clearly linked to B cell function were significantly down regulated in the post treatment samples. Interestingly only one pre-treatment sample had detectable EBV RNA and there were no significant differences in HERV expression (of any group) between pre- and post-treatment samples.Conclusions: While EBV and HERV expression are clearly linked to triggering MS pathogenesis, it does not appear that high level expression of these viruses is a part of the ongoing disease process or that changes in virus load are associated with ocrelizumab treatment

Epstein-Barr virus-associated lymphomas

Epstein–Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host.This article is part of the themed issue ‘Human oncogenic viruses’.</jats:p

SARS-CoV-2 Causes a Different Cytokine Response Compared to Other Cytokine Storm-Causing Respiratory Viruses in Severely Ill Patients

Hyper-induction of pro-inflammatory cytokines, also known as a cytokine storm or cytokine release syndrome (CRS), is one of the key aspects of the currently ongoing SARS-CoV-2 pandemic. This process occurs when a large number of innate and adaptive immune cells activate and start producing pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation. It is one of the factors contributing to the mortality observed with coronavirus 2019 (COVID-19) for a subgroup of patients. CRS is not unique to the SARS-CoV-2 infection; it was prevalent in most of the major human coronavirus and influenza A subtype outbreaks of the past two decades (H5N1, SARS-CoV, MERS-CoV, and H7N9). With a comprehensive literature search, we collected changing the cytokine levels from patients upon infection with the viral pathogens mentioned above. We analyzed published patient data to highlight the conserved and unique cytokine responses caused by these viruses. Our curation indicates that the cytokine response induced by SARS-CoV-2 is different compared to other CRS-causing respiratory viruses, as SARS-CoV-2 does not always induce specific cytokines like other coronaviruses or influenza do, such as IL-2, IL-10, IL-4, or IL-5. Comparing the collated cytokine responses caused by the analyzed viruses highlights a SARS-CoV-2-specific dysregulation of the type-I interferon (IFN) response and its downstream cytokine signatures. The map of responses gathered in this study could help specialists identify interventions that alleviate CRS in different diseases and evaluate whether they could be used in the COVID-19 cases.</p

Unexpected patterns of Epstein-Barr virus transcription revealed by a High throughput PCR array for absolute quantification of viral mRNA

We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein–Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts

The Potential for EBV Vaccines to Prevent Multiple Sclerosis

There is increasing evidence suggesting that Epstein-Barr virus infection is a causative factor of multiple sclerosis (MS). Epstein-Barr virus (EBV) is a human herpesvirus, Human Gammaherpesvirus 4. EBV infection shows two peaks: firstly, during early childhood and, secondly during the teenage years. Approximately, 90-95% of adults have been infected with EBV and for many this will have been a subclinical event. EBV infection can be associated with significant morbidity and mortality; for example, primary infection in older children or adults is the leading cause of infectious mononucleosis (IM). A disrupted immune response either iatrogenically induced or through genetic defects can result in lymphoproliferative disease. Finally, EBV is oncogenic and is associated with several malignancies. For these reasons, vaccination to prevent the damaging aspects of EBV infection is an attractive intervention. No EBV vaccines have been licensed and the prophylactic vaccine furthest along in clinical trials contains the major virus glycoprotein gp350. In a phase 2 study, the vaccine reduced the rate of IM by 78% but did not prevent EBV infection. An EBV vaccine to prevent IM in adolescence or young adulthood is the most likely population-based vaccine strategy to be tested and adopted. National registry studies will need to be done to track the incidence of MS in EBV-vaccinated and unvaccinated people to see an effect of the vaccine on MS. Assessment of vaccine efficacy with MS being a delayed consequence of EBV infection with the average age of onset being approximately 30 years of age represents multiple challenges.</p